Summary: How an organism's genome directs its development and behavior is only beginning to be understood. Knowledge of what genes function in each cell would be a significant step toward a comprehensive understanding. We have developed a system that exploits the invariant lineage of the nematode C. elegans to automatically define gene expression and phenotype throughout development at the single cell level with high temporal resolution. The system uses 2-color, 3D, time-lapse confocal microscopy to record embryogenesis, followed by automated image analysis that identifies and tracks each nucleus through movement, division and death. To date, it can trace the lineage through the first nine out of ten rounds of embryonic cell divisions with high accuracy and assign expression to individual cells. Here, we propose further development of the system. We will extend the system through the last round of cell division. We will also adapt the system for automated phenotypic analysis by measuring various temporal and spatial features of cells and cell groups, as well as changes of gene expression. Furthermore, we will use the methods to systematically decode the function of transcription factors and other genes whose RNAi phenotype is known to be embryonic lethal. Relevance: The completed system will be useful for a wide variety of functional genomics applications and would provide an avenue to comprehensive elucidation of gene function and regulation in C. elegans. Such knowledge in C. elegans would not only provide insights into genes and networks in animal development, but would suggest roles of homologous genes in human health and disease.